ABSTRACT
The use of oligonucleotide-coupled fluorescent microspheres is a rapid, sequencing-independent, and reliable way to diagnose bacterial diseases. Previously described applications of oligonucleotide-coupled fluorescent microspheres for the detection and identification of bacteria in human clinical samples have been successfully adapted to detect and differentiate "Ca. Phytoplasma" species using as a target the chaperonin 60-encoding gene. In this chapter, we describe in detail the design and validation of oligonucleotide capture probes, and their application in the assay aiming to differentiate phytoplasma strains infecting Brassica napus and Camelina sativa plants grown in the same geographic location at the same time.
Subject(s)
In Situ Hybridization/methods , Oligonucleotide Probes , Phytoplasma/genetics , Plant Diseases/microbiology , Brassica napus/genetics , Brassica napus/microbiology , Camellia/genetics , Camellia/microbiology , Chaperonin 60/genetics , DNA, Plant , Fluorescence , Host-Pathogen Interactions , In Situ Hybridization/instrumentation , Microspheres , Oligonucleotide Probes/genetics , Phytoplasma/pathogenicity , Polymerase Chain ReactionABSTRACT
OBJECTIVE: Root canal sealers can interact physically or chemically with dentine. The aim of this study was to characterize the dentine-root canal sealer interface of experimental sealers based on Portland cement using an epoxy-based vehicle in comparison to an epoxy resin sealer, AH Plus. METHODS: Root canals were biomechanically prepared and filled with any one of the four experimental epoxy sealers containing Portland cement with micro- and nano-particles of either zirconium oxide or niobium oxide radiopacifers, or AH Plus. The dentine-sealer's interfaces were assessed by coronal penetration of fluorescent microspheres, the penetration of sealers labelled with Rhodamine B inside the dentine tubules (following obturation with gutta-percha and sealers using System B technique) assessed by confocal laser scanning microscopy, and the chemical characterization of dentine-sealers interface by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) line scans. RESULTS: No penetration of fluorescent microspheres at the root-dentine to sealer interface was recorded for all test materials. Sealers presented greater ability to penetrate within the dentinal tubules at the coronal and mid-root thirds. The experimental sealers containing radiopacifier nano-particles exhibited a more homogeneous microstructure along the whole length of the canal. EDS-line scans results showed a migration of silicon and niobium into dentine. Peak overlap between zirconium and the phosphorous compromised the identification of the migration of the zirconium oxide into dentine. CONCLUSIONS: All five sealers promoted coronal sealing. The experimental sealers exhibited promising characteristics and were comparable to AH Plus sealer. Elemental migration of the experimental sealers suggests material interaction with dentine which was not displayed by AH Plus.